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D-Serine metabolism: spotlight on D-amino acid oxidase - Silvia Sacchi

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Watching at the "D" side: D-amino acids and their significance in neurobiology
June 05 -June 09, 2016 – Lake Como School of Advanced Studies

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D-Serine metabolism: spotlight on D-amino acid oxidase - Silvia Sacchi

  1. 1. Silva Sacchi, Phd Functional  Post-­Genomic and  Protein  Engineering  Lab
  2. 2. Silvia Sacchi 1 Laboratoire de  Neurobiologie Cellulaire et  Moleculaire Institue de  Neiroscience Alfred  Fessard
  3. 3. D-Serine metabolism: spotlight on D-amino acid oxidase Lake  Como  School  of  Advanced  Studies –Summer School Watching at the  «D»  side:  D-­‐amino  acids and  their significancein  neurobiology
  4. 4. DAAO and the flavin cofactor chemistry Oxidized enzyme (yellow) Anionic radical   semiquinone (red) Anionic reduced enzyme (colourless) Silvia Sacchi 1
  5. 5. Silvia Sacchi 2
  6. 6. Silvia Sacchi 3 DAAO catalytic mechanism
  7. 7. Silvia Sacchi 4 DAAO distribution in living organisms Mammals Birds Reptiles & Amphibia Fish Nematodes & Insects Actinobacteria & Actinomycetes Fungi & Yeasts Bacteria
  8. 8. Silvia Sacchi 5 DAAO distribution in living organisms
  9. 9. DAAO in mammals Silvia Sacchi 6
  10. 10. Silvia Sacchi 7 DAAO in mammals D-­‐AMINO  ACIDS  ELIMINATION OXALATE  CONCENTRATION   REGULATION INVOLVED  IN  AN  ANTIBACTERIAL  SYSTEM  IN   NEUTROPHILIC  LEUKOCYTES
  11. 11. Silvia Sacchi 8 DAAO distribution in the brain D-serine DAAO activity DAAO expression DAAO activity DAAO DAAO DAAO DAAO DAAO DAAO DAAO
  12. 12. To keep in mind: D-aspartate oxidase is also present in mammalian brain Silvia Sacchi 9 ▲ D-proline (usually used as a substrate in activity assays) is also catabolized by DDO ▲ The specificty of the signals detected during immunolocalization studies has to be checked
  13. 13. The «serine shuttle» model and DAAO role Silvia Sacchi 10
  14. 14. Silvia Sacchi 11 1) DAAOs biochemical properties 2) Regulation of hDAAO activity
  15. 15. Silvia Sacchi 12 DAAOs biochemical properties ► stabilize the anionic semiquinone form of the cofactor ► covalently bind sulfite ► the oxidized forms are fully converted to the reduced forms upon the addition of the substrate in anaerobic conditions ► the reduced enzymes are highly reactive with molecular oxygen
  16. 16. Silvia Sacchi 13 ►Oligomeric state DAAOs biochemical properties Holoenzyme Apoprotein human pig rat yeast dimeric dependent on  [DAAO] monomeric dimeric/monomeric dimeric monomeric monomeric monomeric DAAO From:  Kawazoe et al,  Protein Science  (2006),  15:2708–2717 pighumanFrom:  Caldinelli et al,  ProteinScience  (2009),  18:801–810
  17. 17. Silvia Sacchi 14 ►Oligomeric state DAAOs biochemical properties Holoenzyme Apoprotein human pig rat yeast dimeric dependent on  [DAAO] monomeric dimeric/monomeric dimeric monomeric monomeric monomeric DAAO rDAAO hDAAO Arg120 Gln234 His78
  18. 18. Silvia Sacchi 15 Holoenzyme Tm (°C) human pig rat yeast dimeric dependent on  [DAAO] monomeric dimeric/monomeric 57 n.d. 47 50 DAAO The monomeric state yields to a less stable enzyme From:  Frattini  et  al,  FEBS  Journal  (2011)  278:4362–4373
  19. 19. Silvia Sacchi 16 ►FAD binding DAAOs biochemical properties + Benz 0.03 0.2
  20. 20. Silvia Sacchi 17
  21. 21. Silvia Sacchi 18
  22. 22. Silvia Sacchi 19 ►Ligand binding DAAOs biochemical properties 5-­Chlorobenzo[d]isoxazol-­3-­ol CBIO
  23. 23. Silvia Sacchi 20 ►Kinetic & substrate specificity DAAOs biochemical properties Best substrates D-DOPA D-Tyr D-Ala D-Pro D-Trp D-Phe D-Pro D-Met D-Ala D-Phe D-Trp D-Ala
  24. 24. Does hDAAO really exert the proposed physiological role ? Silvia Sacchi 21 From:  Morikawa et  al.,  J.  Chromatogr.   B  (2001)  757:119  –125
  25. 25. Silvia Sacchi 22 LTP in ddY/DAAO-/- mice From:  Maekawa et  al.,  Neuroscience Research(2005)  53:34–38
  26. 26. Silvia Sacchi 23
  27. 27. Silvia Sacchi 24 From:  Hashimoto et al,  Arch Psychiatry ClinNeurosci (2013)  263:367-­‐377 Schizophrenia – the glutamatergic hypothesis From: Harrison et al, Molecular Psychiatry (2005) 10:40-68
  28. 28. Silvia Sacchi 25 Schizophrenia– alternative treatment acting on NMDAR From:  Hashimoto et al,  Arch Psychiatry ClinNeurosci (2013)  263:367-­‐377
  29. 29. Silvia Sacchi 26 1) DAAOs biochemical properties 2) Regulation of hDAAO activity ►inhibitors ►mutations ►interacting protein Key features: ► very low Kd, Ki and/or IC50 ► ability to reach the molecular target in vivo ► stabiltity in biological fluids/tissues
  30. 30. Silvia Sacchi 27 hDAAO inhibitors From:  Sacchi  et al,  Curr Pharm Des (2013)  19:2499-­‐2511 Kd (µM) IC50 (µM) human rat pig 7 150 2 189 Benzoate Benzoate  produced  a  21%  improvement   in  Positive  and  Negative  Syndrome   Scale  total  score
  31. 31. Silvia Sacchi 28 hDAAO inhibitors From:  Sacchi  et al,  Curr Pharm Des (2013)  19:2499-­‐2511 Kd (µM) IC50 (µM) human rat pig 7 150 2 189 Benzoate From: Ferraris et al, Curr Pharm Des (2011) 17:103-111 1 2Benzoate 1=  4H-­‐furo[3,2-­‐b]pyrrole-­‐5-­‐carboxylic  acid 2=  3-­‐hydroxyquinolin-­‐2-­‐(1H)-­‐one Kd (nM)   IC50 (nM) 1 2 3,1 13 141 4 hDAAO
  32. 32. Silvia Sacchi 29 From: Hopkins et al, J Medical Chem (2013) 56:3710-3724 Microdialysis studies  in  rat  cerebellum Rats  treated  with  NMDAR  antagonist  L-­‐701,324
  33. 33. hDAAO «second generation» inhibitors From:  Hondo et al,  J  Medical Chem (2013)  56:3582-­‐3592 Silvia Sacchi 30
  34. 34. Silvia Sacchi 31 From:  Terry-­‐Lorenzo  et  al,  Bioscience Report  (2014)  11;34(4)
  35. 35. Silvia Sacchi 32 1) DAAOs biochemical properties 2) Regulation of hDAAO activity ►inhibitors ►mutations ►interacting protein activating inactivatinginactivating
  36. 36. Silvia Sacchi 33 hDAAO inactivating mutations R199 substitutions modify hDAAO conformation ► the tertiary structure of the variants is altered R199W R199Q
  37. 37. Silvia Sacchi 34 hDAAO inactivating mutations R199 substitutions modify hDAAO conformation ► the tertiary structure of the variants is altered ► the variants show a modified oligomeric state ► the melting temperature is altered
  38. 38. Silvia Sacchi 35
  39. 39. Silvia Sacchi 36
  40. 40. 1) DAAOs biochemical properties 2) Regulation of hDAAO activity ►inhibitors ►mutations ►interacting protein Silvia Sacchi 37 activating inactivating activating Trp209 Asp31 Arg279 Trp209 Asp31 Arg279 Asp31His Trp209Arg Arg279Ala
  41. 41. Silvia Sacchi 38 hDAAO activating mutations
  42. 42. Silvia Sacchi 39 1) DAAOs biochemical properties 2) Regulation of hDAAO activity From:  Popiolek et  al,  J  Biol Chem (2011)  286:28867-­‐28875 ►inhibitors ►mutations ►interacting protein DAAO might be localized also otuside peroxisomes, exposed to a larger set of interacting protein!
  43. 43. Silvia Sacchi 40 Modulation of hDAAO functionality by interacting proteins DAAO-BSN interaction scenario: At the presynaptic active zone DAAO is inhibited by the colocalizing BSN and this concurs to the correct fuctioning of the synapses
  44. 44. Silvia Sacchi 41 Modulation of hDAAO functionality by interacting proteins G72  encodes for pLG72  (153  AA) ü unknown  function ü primate  specific  gene  -­‐ chromosome  13 ü expressed  in  spinal  cord,  testis  and  brain ü subcellular localization: mitochondria Gel filtrationQuantitative IP BIACORE Plasmon Surface Resonance Kd ≈  10-­‐6 – 10-­‐7 M
  45. 45. Silvia Sacchi 42 pLG72 modulates hDAAO activity pLG72 acts as an inactivator of hDAAO
  46. 46. Silvia Sacchi 43 hDAAO and pLG72 interact in U87 transfected cells Peroxisomal Mitochondrial U87 pLG72-ECFP cells transfeced with EYFP-hDAAO From:  Sacchi  et  al,  Mol Cell  Neurosc (2011)  48:20-­‐28
  47. 47. Cytosolic hDAAO is active Silvia Sacchi 44 From:  Sacchii et  al,  Mol Cell  Neurosc (2011)  48:20-­‐28
  48. 48. Silvia Sacchi 45 hDAAO and pLG72 interact in U87 transfected cells U87 pLG72-ECFP cells transfected with EYFP-hDAAO From:  Sacchi  et  al,  Mol Cell  Neurosc (2011)  48:20-­‐28
  49. 49. Silvia Sacchi 46
  50. 50. Silvia Sacchi 47 pLG72 affects hDAAO stability From:  Cappelletti  et  al,  Mol Cell  Neurosc (2011)  48:20-­‐28 ™ CHX treatment and WB analysis ¢ pulse-chase experiment hDAAO is a stable protein within the cell, while pLG72 is a short-lived protein
  51. 51. Silvia Sacchi 48 Structural details of hDAAo-pLG72 interaction
  52. 52. Silvia Sacchi 49 Structural details of hDAAO-pLG72 interaction Limited  proteolysis  experiments preferential proteolyticsites: C  =chymotrypsin;  S  =  subtilisin;  T  =  trypsin; G  =  endoproteaseGlu-­‐C;  X  =  SB3  cross-­‐link  (red bar) preferential proteolyticsites: blue  =  hDAAO alone purple  =  pLG72  complexed  hDAAO Red  =  chemical  cross-­‐link
  53. 53. Silvia Sacchi 50 Structural details of hDAAO-pLG72 interaction Cross-­‐linking  experiments   BS3 bis(sulfosuccinimidyl)suberate overall  identity  =  10.3% maximal  identity  =  27% (considering  only  ∼ 30%  of  the  template)
  54. 54. A starting point: pLG72 structure model Silvia Sacchi 51 α-­‐helix  content  : model,  predicted  =  62% experimental  =  73%
  55. 55. Silvia Sacchi 52 overall  identity  =  10.3% maximal  identity  =  27% (considering  only  ∼ 30%  of  the  template)
  56. 56. Silvia Sacchi 53 hDAAO-pLG72 complex structure model Note:  Solvent-­‐accessible  surface  calculations  point  to  a  stable  interaction
  57. 57. Silvia Sacchi 54 pLG72 truncated forms deleted  in pLG72  1-­‐123 deleted  in pLG72  72-­‐153
  58. 58. Silvia Sacchi 55 Properties of pLG72 deleted variants ►Accumulated as inclusion bodies when expressed in E. coli ►Similar content of secondary structure elements ►Dimeric ►Bind large aromatic compounds (CPZ) ►Stable to thermal unfolding 1-­‐153 1-­‐123 1-­‐94 72-­‐153 1-­‐64 35075±315 33818±461 28307±56 24815±175 20707±82 pLG72 variants Mol.  Mass by  DLS  (Da) 18079 17576 14162 12743 10526 Mol.  Mass (Da) 1,6±0,2 0,8±0,3 0,9±0,3 2,3±0,3 1,0±0,3 Kd CPZ (µM) 59,2±2,7 66,3±11,0 50,9±4,6 64,6±1,2 73,7±1,4 Tm (°C)
  59. 59. pLG72 deleted variants bind hDAAO, Silvia Sacchi 56 SPR  analysis hDAAOhDAAOhDAAO pLG72 CHIP pLG72 flow
  60. 60. … but inhibited the flavoenzyme to a limited extent Silvia Sacchi 57 Activity  assays  after  30  min  incubation  – different  hDAAO:pLG72  variants  molar  ratio 80% 25-­‐50% inhibition
  61. 61. Silvia Sacchi 58 Structural details of hDAAO-pLG72 interaction deleted  in pLG72  1-­‐123 deleted  in pLG72  72-­‐153 C-terminal region regulation of protein conformation & hDAAO interaction N-terminal region regulation of hDAAO binding
  62. 62. Silvia Sacchi 59 hDAAO inhibitors, new classes of compounds From:  Terry-­‐Lorenzo  et  al,  J  Biomol Screening(2015)  20:1218-­‐1231
  63. 63. Conclusions 1. hDAAO shows peculiar biochemical properties 3. hDAAO functionality can be modulated 2. hDAAO has an important role in controlling D-serine cellular levels Regulatory protein – pLG72 ► interacts with hDAAO before it is targeted to peroxisimes ► acts as a «negative chaperone» Silvia Sacchi 60
  64. 64. Future perspective & issues Unravelling the molecular mechanisms of hDAAO functionality regulation Designing novel strategies to regulate NMDAr activation state Promote novel therapeutic approaches Silvia Sacchi 61
  65. 65. Silvia Sacchi 62 Aknowledgements Ryan  Terry-­‐Lorenzo Seth  Hopkins Leila  Birolo Piero  Pucci Loredano Pollegioni Gianluca  Molla Pamela  Cappelletti Laura  Caldinelli Jean-­‐Pierre Mothet Magalie Martineau Emila  Pedone Luciano  Pirone Giovanni  Smaldone
  66. 66. Silvia Sacchi 63 Aknowledgements

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